The cellular gene c-src and its viral homologue v-src (the transforming gene of Rous sarcoma virus) encode 60 kDa, cytoplasmic, membrane- associated, protein-tyrosine kinases. For the viral kinase or for transforming mutants of the cellular kinase, a close correlation exists between elevated specific activity and cell transformation. The overall goal of this proposal is to define molecular mechanisms that regulate the Src kinases in normal cells, and those that deregulate them in cancer cells. Our hypothesis is that specific domains of Src, and the proteins that bind to them, are important regulators of kinase activity. I propose to isolate, identify and characterize proteins that bind to Src and regulate its activity. I will use the yeast two-hybrid system to screen human fibroblast and colon carcinoma cDNA libraries for proteins which interact with the unique domain (UD), UD/SH3, UD/SH3/SH2, SH3/SH2, SH3 or SH2 region of Src. The site on Src required for binding to an interacting protein will be determined, mutations will be introduced into the binding site and the functional consequences on Src activity will be assessed. These studies should increase our understanding of signaling via oncogenic tyrosine kinases.